An RNA-binding domain in the viral haemorrhagic septicaemia virus nucleoprotein.
The gene encoding the nucleoprotein (N) and PCR-derived subfragments from viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were overexpressed in Escherichia coli BL21(DE3) transformed by recombinant expression vector pET-14b containing N and PCR-generated sub-fragment cDNAs under the control of the T7 RNA polymerase promoter. Following induction with IPTG, recombinant His-tagged proteins were expressed, purified by affinity metal chelation chromatography under denaturing conditions and renatured. Protein blots were hybridized with various radiolabelled nucleic acid probes. Results obtained using genomic or messenger virus RNA as a probe indicated that the middle part of N was possibly an RNA-binding domain. To confirm this observation, two more accurate approaches were undertaken: (i) a gel retardation assay of RNA and purified protein complexes was done; and (ii) RNA-protein complexes were cross-linked by UV light and analysed on a denaturing polyacrylamide gel. All these experiments led us to conclude that the middle part of N is the domain which interacts with RNA, despite the absence of homology with known consensus amino acid sequences of other RNA-binding proteins.
Said
T
T
Unité de Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, jouy-en-Josas, France.
Bruley
H
H
Lamoureux
A
A
Brémont
M
M
D001665:Binding Sites; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D015347:RNA Probes / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012209:Rhabdoviridae / Q000378:metabolism*
D009698:Nucleoproteins; D015347:RNA Probes; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012313:RNA